Understanding how phospholipids orchestrate cellular signaling requires tools that preserve their native complexity to reveal their modular function. Here, we introduce a versatile chemical approach to map the phosphatidic acid (PA) interactome directly within membranes of living cells. Using synthetic PA analogues, modified on the glycerol backbone with a bioorthogonal azide group, we generated functional probes that retain natural headgroup and acyl-chain composition and properties. These analogues can be coupled to photoactivatable crosslinkers, allowing covalent capture of transient PA-protein partners upon UV illumination. We present here approaches to validate their integration and functionality through recruitment assays using PA-binding sensor and ERK1/2 phosphorylation readouts. Applying this framework in neurosecretory PC12 cells, we identified specific PA-binding partners across distinct secretory states, revealing both acyl chain-dependent and activity-specific interaction patterns. This methodology bridges lipid chemistry and cell biology by enabling dynamic, species-specific exploration of phospholipid signaling networks. Beyond neurosecretion, the approach provides a broadly applicable platform for dissecting lipid-mediated processes across diverse physiological and pathological contexts, from membrane trafficking to disease-associated lipid signaling.

The cell plasma membrane acts as a semi-permeable barrier essential for cellular protection and function, posing a challenge for therapeutic molecule delivery. Conventional techniques for crossing this barrier, including biophysical and biochemical methods, often exhibit limitations such as cytotoxicity and the risk of genomic integration when viral vectors are involved. In contrast, cell-penetrating peptides (CPPs) offer a promising non-invasive means to deliver a broad range of molecular cargoes, including proteins, nucleic acids and small molecules, into cells. CPPs, typically 5 to 30 amino acids long and rich in basic or non-polar residues, interact favourably with different cell membranes. These peptides have evolved since the discovery of the HIV-1 TAT peptide in the 1980s, expanding into various CPP families with diverse therapeutic applications. CPPs can form covalent or non-covalent complexes with their cargo, influencing their stability and efficacy. Based on their sequence properties and interactions, CPPs can be amphipathic or non-amphipathic, with distinct mechanisms of membrane penetration, such as direct penetration and endocytosis. While their uptake mechanisms are complex and not fully elucidated, ongoing optimization aims to enhance CPP specificity and efficacy. CPPs have demonstrated potential in drug delivery, gene therapy, cancer treatment and vaccine development, addressing key safety and efficiency concerns associated with viral vectors. This review explores the classification, mechanisms of action and therapeutic potential. It focuses on the intracellular vesicular trafficking of CPPs, highlighting their role as transformative tools in advancing cellular therapies and medical treatments.

Glycerophospholipids (GPLs) play important roles in cellular compartmentalization and signaling. Among them, phosphatidic acids (PA) exist as many distinct species depending on acyl chain composition, each one potentially displaying unique signaling function. Although the signaling functions of PA have already been demonstrated in multiple cellular processes, the specific roles of individual PA species remain obscure due to a lack of appropriate tools. Indeed, current synthetic PA analogues fail to preserve all the functions of natural PA. To circumvent these limitations, we developed a novel synthetic approach to produce PA analogues without compromising structural integrity of acyl chains. Moreover, addition of a clickable moiety allowed flexible grafting of different molecules to PA analogues for various biological applications. Hence, this innovation also provides powerful tools to investigate specific biological activities of individual PA species, with potential applications in unraveling complex GPL-mediated signaling pathways.

Neuroendocrine tumors (NETs) constitute a heterogeneous group of neoplasms arising from hormone-releasing cells. Secretion of hormones stored in vesicles occurs through calcium-regulated exocytosis, a process that needs to be tightly controlled to avoid unbalanced levels of hormones. A critical feature shared by most of the NETs is a dysfunctional secretory pathway mainly leading to hypersecretion, which often induces clinical complications. In this review, we focus on the cellular process of hormone exocytosis and discuss the potential molecular mechanisms leading to deregulated hormone secretion in various NETs. Particular attention is paid to expression level modifications for genes and proteins involved in the exocytic pathway in NETs.

The plasma membrane of eukaryotic cells is highly dynamic and asymmetrically organized. Its continuous remodeling plays a crucial role in diverse cellular processes, including apoptosis, blood coagulation, and vesicular trafficking. The distribution and rearrangement of phospholipids (PLs) within the bilayer are tightly regulated, influencing membrane curvature, tension, and organization. This review examines the role of PL asymmetry in vesicle fusion, the final step of exocytosis, and in vesicular membrane retrieval by compensatory endocytosis in neurosecretory cells, with a particular emphasis on structural PLs such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). We discuss the molecular mechanisms that maintain and disrupt PLs asymmetry and explore how lipid rearrangements affect vesicle dynamics. Additionally, we highlight recent findings on lipid scramblases, particularly phospholipid scramblase-1 (PLSCR1), and their role in regulated exocytosis and compensatory endocytosis.

CPPs, or Cell-Penetrating Peptides, offer invaluable utility in disease treatment due to their ability to transport various therapeutic molecules across cellular membranes. Their unique characteristics, such as biocompatibility and low immunogenicity, make them ideal candidates for delivering drugs, genes, or imaging agents directly into cells. This targeted delivery enhances treatment efficacy while minimizing systemic side effects. CPPs exhibit versatility, crossing biological barriers and reaching intracellular targets that conventional drugs struggle to access. This capability holds promise in treating a wide array of diseases, including cancer, neurodegenerative disorders, and infectious diseases, offering a potent avenue for innovative and targeted therapies, yet their precise mechanism of cell entry is far from being fully understood. In order to correct Cu dysregulation found in various pathologies such as Alzheimer disease, we have recently conceived a peptide Cu(II) shuttle, based on the αR5W4 CPP, which, when bound to Cu(II), is able to readily enter a neurosecretory cell model, and release bioavailable Cu in cells. Furthermore, this shuttle has the capacity to protect cells in culture against oxidative stress-induced damage which occurs when Cu binds to the Aβ peptide. The aim of this study was therefore to characterize the cell entry route used by this shuttle and determine in which compartment Cu is released. Pharmacological treatments, siRNA silencing and colocalization experiments with GFP-Rab fusion proteins, indicate that the shuttle is internalized by an ATP-dependent endocytosis pathway involving both Rab5 and Rab14 endosomes route and suggest an early release of Cu from the shuttle.

Phospholipids (PLs) are asymmetrically distributed at the plasma membrane. This asymmetric lipid distribution is transiently altered during calcium-regulated exocytosis, but the impact of this transient remodeling on presynaptic function is currently unknown. As phospholipid scramblase 1 (PLSCR1) randomizes PL distribution between the two leaflets of the plasma membrane in response to calcium activation, we set out to determine its role in neurotransmission. We report here that PLSCR1 is expressed in cerebellar granule cells (GrCs) and that PLSCR1-dependent phosphatidylserine egress occurred at synapses in response to neuron stimulation. Synaptic transmission is impaired at GrC Plscr1(-/-) synapses, and both PS egress and synaptic vesicle (SV) endocytosis are inhibited in Plscr1(,) cultured neurons from male and female mice, demonstrating that PLSCR1 controls PL asymmetry remodeling and SV retrieval following neurotransmitter release. Altogether, our data reveal a novel key role for PLSCR1 in SV recycling and provide the first evidence that PL scrambling at the plasma membrane is a prerequisite for optimal presynaptic performance.

Structural phospholipids are asymmetrically distributed at the plasma membrane, with phosphatidylethanolamine and phosphatidylserine (PS) virtually absent from the outer leaflet. This asymmetric lipid distribution is transiently altered during specific biological processes including calcium-regulated exocytosis. However, the impact of this transient remodeling of membrane asymmetry on presynaptic function remains unknown. PhosphoLipid SCRamblase 1 (PLSCR1), a protein that randomizes phospholipid distribution between the two leaflets of the plasma membrane in response to calcium activation is an ideal candidate to alter this asymmetry. We therefore set out to determine the role of PLSCR1 in both neurotransmitter release and synaptic vesicle recycling by combining electron microscopy, optical live cell imaging of pHluorin probes and electrophysiology in cerebellar granule cells (GrC) from Plscr1 knock-out mice ( Plscr1 -/- ). We report that PLSCR1 is expressed in GrCs and that PLSCR1-dependent PS egress occurred at synapses in response to neuron stimulation. Furthermore, synaptic transmission is impaired at GrC Plscr1 -/- synapses and both PS egress and synaptic vesicle endocytosis are inhibited in Plscr1 -/- cultured neurons, demonstrating that PLSCR1 controls phospholipid asymmetry remodeling and synaptic vesicle retrieval following neurotransmitter release. Altogether, our data reveal a key role for PLSCR1 in synaptic vesicle recycling and provide the first evidence that phospholipid scrambling at the plasma membrane is a prerequisite for optimal presynaptic performance.

Despite advances in Electron Microscopy (EM) that enable to image protein assemblies within vitreous sections of cells at nearly atomic resolution, labelling is still necessary to locate small proteins or rare complexes. Gold immunolabelling has been used for decades to localise specific proteins within cellular sections. However, current gold particle-antibody conjugates are not built with enough chemical precision to match the current resolution offered by cryo-EM methodology. Furthermore, as a close to native specimen state can only be achieved by strict preservation of a frozen hydrated state, it is required to deliver gold labelling agents into living cells prior to their vitrification. Several 1.4 nm gold nanoparticle-antibody conjugates were synthesised. Their abilities to bind to and label their corresponding epitopes within living cells after cytosolic delivery by electroporation are documented here.

Although there is mounting evidence indicating that lipids serve crucial functions in cells and are implicated in a growing number of human diseases, their precise roles remain largely unknown. This is particularly true in the case of neurosecretion, where fusion with the plasma membrane of specific membrane organelles is essential. Yet, little attention has been given to the role of lipids. Recent groundbreaking research has emphasized the critical role of lipid localization at exocytotic sites and validated the essentiality of fusogenic lipids, such as phospholipase D (PLD)-generated phosphatidic acid (PA), during membrane fusion. Nevertheless, the regulatory mechanisms synchronizing the synthesis of these key lipids and neurosecretion remain poorly understood. The vacuolar ATPase (V-ATPase) has been involved both in vesicle neurotransmitter loading and in vesicle fusion. Thus, it represents an ideal candidate to regulate the fusogenic status of secretory vesicles according to their replenishment state. Indeed, the cytosolic V1 and vesicular membrane-associated V0 subdomains of V-ATPase were shown to dissociate during the stimulation of neurosecretory cells. This allows the subunits of the vesicular V0 to interact with different proteins of the secretory machinery. Here, we show that V0a1 interacts with the Arf nucleotide-binding site opener (ARNO) and promotes the activation of the Arf6 GTPase during the exocytosis in neuroendocrine cells. When the interaction between V0a1 and ARNO was disrupted, it resulted in the inhibition of PLD activation, synthesis of phosphatidic acid during exocytosis, and changes in the timing of fusion events. These findings indicate that the separation of V1 from V0 could function as a signal to initiate the ARNO-Arf6-PLD1 pathway and facilitate the production of phosphatidic acid, which is essential for effective exocytosis in neuroendocrine cells.

Neuroendocrine tumors constitute a heterogeneous group of tumors arising from hormone-secreting cells and are generally associated with a dysfunction of secretion. Pheochromocytoma (Pheo) is a neuroendocrine tumor that develops from chromaffin cells of the adrenal medulla, and is responsible for an excess of catecholamine secretion leading to severe clinical symptoms such as hypertension, elevated stroke risk and various cardiovascular complications. Surprisingly, while the hypersecretory activity of Pheo is well known to pathologists and clinicians, it has never been carefully explored at the cellular and molecular levels. In the present study, we have combined catecholamine secretion measurement by carbon fiber amperometry on human tumor cells directly cultured from freshly resected Pheos, with the analysis by mass spectrometry of the exocytotic proteins differentially expressed between the tumor and the matched adjacent non-tumor tissue. In most patients, catecholamine secretion recordings from single Pheo cells revealed a higher number of exocytic events per cell associated with faster kinetic parameters. Accordingly, we unravel significant tumor-associated modifications in the expression of key proteins involved in different steps of the calcium-regulated exocytic pathway. Altogether, our findings indicate that dysfunction of the calcium-regulated exocytosis at the level of individual Pheo cell is a cause of the tumor-associated hypersecretion of catecholamines.

Glass beads of the Sr2MgSi2O7 stoichiometric composition and a non-stoichiometric composition with higher SiO2/SrO ratio doped with Eu2O3/Dy2O3 were prepared through aerodynamic levitation coupled to CO2 laser heating. The glass beads were subsequently treated at 1100 degrees C to produce glass-ceramics with Sr2MgSi2O7: Eu2+, Dy3+ as the main crystalline phase. The doped glasses exhibit red emissions; after crystallisation, the corre-sponding glass-ceramics emit blue light under UV excitation. The starting glass composition considerably affects the crystallisation process, resulting in Sr2MgSi2O7 glass-ceramics with very different microstructures which, in turn, have a significant influence on the luminescence properties. The photoluminescence emission spectra of the glass-ceramics under UV light show a broadband emission (lambda = 400-500 nm) with a main peak assigned to the typical Eu2+ transition under excitation at 365 nm. Both the intensity of the emission and the persistence time significatively increase on decreasing temperature. Glass-ceramics from the non-stoichimetric glass composition co-doped with 1Eu2O3/0.5Dy2O3 (mol%.) provided the longest persistence times.

The synthetic peptide AKH-αR5W4 NBD was designed as a shuttle to counteract copper imbalance in Alzheimer’s disease. In vitro , this shuttle is able to abstract Cu( ii ) selectively from amyloid-β and transport it into cells in a bioavailable form.

Increasingly common, neuroendocrine tumors (NETs) are regarded nowadays as neoplasms potentially causing debilitating symptoms and life-threatening medical conditions. Pheochromocytoma is a NET that develops from chromaffin cells of the adrenal medulla, and is responsible for an excessive secretion of catecholamines. Consequently, patients have an increased risk for clinical symptoms such as hypertension, elevated stroke risk and various cardiovascular complications. Somatostatin analogues are among the main anti-secretory medical drugs used in current clinical practice in patients with NETs. However, their impact on pheochromocytoma-associated catecholamine hypersecretion remains incompletely explored. This study investigated the potential efficacy of octreotide and pasireotide (SOM230) on human tumor cells directly cultured from freshly resected pheochromocytomas using an implemented catecholamine secretion measurement by carbon fiber amperometry. SOM230 treatment efficiently inhibited nicotine-induced catecholamine secretion both in bovine chromaffin cells and in human tumor cells whereas octreotide had no effect. Moreover, SOM230 specifically decreased the number of exocytic events by impairing the stimulation-evoked calcium influx as well as the nicotinic receptor-activated inward current in human pheochromocytoma cells. Altogether, our findings indicate that SOM230 acts as an inhibitor of catecholamine secretion through a mechanism involving the nicotinic receptor and might be considered as a potential anti-secretory treatment for patients with pheochromocytoma.

Metabolic health depends on the brain's ability to control food intake and nutrient use versus storage, processes that require peripheral signals such as the adipocyte-derived hormone, leptin, to cross brain barriers and mobilize regulatory circuits. We have previously shown that hypothalamic tanycytes shuttle leptin into the brain to reach target neurons. Here, using multiple complementary models, we show that tanycytes express functional leptin receptor (LepR), respond to leptin by triggering Ca2+ waves and target protein phosphorylation, and that their transcytotic transport of leptin requires the activation of a LepR-EGFR complex by leptin and EGF sequentially. Selective deletion of LepR in tanycytes blocks leptin entry into the brain, inducing not only increased food intake and lipogenesis but also glucose intolerance through attenuated insulin secretion by pancreatic β-cells, possibly via altered sympathetic nervous tone. Tanycytic LepRb-EGFR-mediated transport of leptin could thus be crucial to the pathophysiology of diabetes in addition to obesity, with therapeutic implications.

Calcium-regulated exocytosis is a multi-step process that allows specialized secretory cells to release informative molecules such as neurotransmitters, neuropeptides, and hormones for intercellular communication. The biogenesis of secretory vesicles from the Golgi cisternae is followed by their transport towards the cell periphery and their docking and fusion to the exocytic sites of the plasma membrane allowing release of vesicular content. Subsequent compensatory endocytosis of the protein and lipidic constituents of the vesicles maintains cell homeostasis. Despite the fact that lipids represent the majority of membrane constituents, little is known about their contribution to these processes. Using a combination of electrochemical measurement of single chromaffin cell catecholamine secretion and electron microscopy of roof-top membrane sheets associated with genetic, silencing and pharmacological approaches, we recently reported that diverse phosphatidic acid (PA) species regulates catecholamine release efficiency by controlling granule docking and fusion kinetics. The enzyme phospholipase D1 (PLD1), producing PA from phosphatidylcholine, seems to be the major responsible of these effects in this model. Here, we extended this work using spinning disk confocal microscopy showing that inhibition of PLD activity also reduced the velocity of granules undergoing a directed motion. Furthermore, a dopamine β-hydroxylase (DβH) internalization assay revealed that PA produced by PLD is required for an optimal recovery of vesicular membrane content by compensatory endocytosis. Thus, among numerous roles that have been attributed to PA our work gives core to the key regulatory role in secretion that has been proposed in different cell models. Few leads to explain these multiple functions of PA along the secretory pathway are discussed.

Pheochromocytoma (Pheo) is a neuroendocrine tumor that develops from chromaffin cells of the adrenal medulla, and is responsible of an excess of catecholamines secretion leading to severe clinical symptoms such as hypertension, elevated stroke risk and various cardiovascular complications. Surprisingly, hypersecretory activity of Pheo has never been explored at the cellular and molecular levels from individual tumor cells. In the present study, we have combined catecholamine secretion measurement by carbon fiber amperometry on human tumor cells directly cultured from freshly resected Pheo, with the analysis by mass spectrometry of the exocytotic proteins differentially expressed between the tumor and the matched adjacent non-tumor tissue. Catecholamine secretion recordings from individual Pheo cells obtained from most patients reveal a higher number of exocytic events per cell associated with faster kinetic parameters. Accordingly, we unravel significant tumor-associated modifications in the expression of key proteins involved in different steps of the calcium-regulated exocytic pathway. Altogether, our findings indicate that dysfunction of the calcium-regulated exocytosis at the level of individual Pheo cell is a cause of the tumor-associated hypersecretion of catecholamines.